(1) Field of the Invention
The present invention generally relates to enzyme inhibitors. More specifically, the invention relates to methods of designing transition state inhibitors of a 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase
(2) Description of the Related Art
References cited
Allart, B., Gatel, M., Guillerm, D., and Guillerm, G. (1998) The catalytic mechanism of adenosylhomocysteine/methylthioadenosine nucleosidase from Escherichia coli—chemical evidence for a transition state with a substantial oxocarbenium character. Eur. J. Biochem. 256, 155-162.
Anet, F. A. L.; Basus, V. J.; Hewett, A. P. W.; Saunders, M. (1980) J. Am. Chem. Soc. 102, 3945-3946.
Anisimov, V., and Paneth, P. (1999) ISOEFF98. A program for studies of isotope effects using Hessian modifications, J. Math. Chem. 26, 75-86.
Bagdassarian, C. K., Schramm, V. L., and Schwartz, S. D. (1996) Molecular electrostatic potential analysis for enzymatic substrates, competitive inhibitors and transition-state inhibitors, J. Am. Chem. Soc. 118, 8825-8836.
Becke, D. A. (1996) Density-functional thermochemistry. IV. A new dynamical correlation functional and implications for exact-exchange mixing J. Chem. Phys. 104, 1040-1046.
Bennet, A. J.; Sinnot, M. L. (1986) J. Am. Chem. Soc. 108, 7287-7294
Berti, P. J., and Tanaka K. S. E. (2002) Transition state analysis using multiple kinetic isotope effects: Mechanisms of enzymatic and non-enzymatiC glycoside hydrolysis and transfer. Adv. Phys. Org. Chem. 37, 239-314.
Birck, M., and Schramm, V. L., (2004a) Nucleophilic participation in the transition state for human thymidine phosphorylase. J. Am. Chem. Soc. 126, 2447-2453.
Birck, M. R., and Schramm, V. L. (2004b) Binding Causes the remote [5′-3H]thymidine kinetic isotope effect in human thymidine phosphorylase, J. Am. Chem. Soc. 126, 6882-6883.
Cadieux, N.; Bradbeer, C.; Reeger-Schneider, E.; Koster, W.; Mohanty, A. K.; Wiener, M. C.; Kadner, R. J. (2002) J. Bacteriol. 184, 706-717.
Carteni-Farina, M., Porcelli, M., Cacciapuoti, G., Zappia, V., Grieko, M., and Difiore P. P. (1983) Adv. Polyamine Res. 4, 779-792.
Cha, Y.; Murray, C. J.; Klinman, J. P. (1989) Science 243, 1325-1330.
Chen, X. Y., Berti, P. J., and Schramm, V. L. (2000) Ricin A-Chain: Kinetic Isotope Effects and Transition State Structures with Stem-Loop RNA, J. Am. Chem. Soc. 122, 1609-1617.
Chen, X., Schauder, S., Potier, N., Dorsselaer, V. A., Pelczer, I., Bassler, B. L., and Hughson, F. M. (2002) Structural identification of a bacterial quorum-sensing signal containing boron, Nature 415, 545-549.
Cleland, W. W. (2005) Arch. Biochem. Biophy. 433, 2-12.
Cornell, K. A., Swarts, W. E., Barry, R. D., and/Riscoe, M. K. (1996) Characterization of recombinant Eschericha coli 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase: analysis of enzymatic activity and substrate specificity, Biochem. Biophys. Res. Commun. 228, 724-732.
Craig, B. N.; Janssen, M. U.; Wickersham, B. M.; Rabb, D. M.; Chang, P. S.; O'Leary, D. J. (1996) J. Org. Chem. 61, 9610-9613.
Cramer, C. J.; Truhlar, D. J. (1999) Chem. Rev. 99, 2161-2200.
DeWolf, W. E., Jr., Fullin, F. A., and Schramm, V. L. (1979) The catalytic site of AMP nucleosidase. Substrate specificity and pH effects with AMP and formycin 5′-PO4, J. Biol. Chem. 254, 10868-10875.
Dwyer, J. J.; Gittis, A. G.; Karp, D. A.; Lattman, E. E.; Spencer, D. S.; Stities, W. E.; Garcia-Moreno, B. E. (2000) Biophysical Journal 79, 1610-1620.
Flükiger, P., Lüthi, H. P., Portmann, S., and Weber, J. (2000) MOLEKEL 4.0, Swiss Center for Scientific Computing, Manno, Switzerland.
Frisch, M. J., G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. A. Montgomery, Jr., T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M. ada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J. mDannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong, C. Gonzalez, and J. A. Pople, Gaussian 03, Revision B.04, Gaussian, Inc., Pittsburgh Pa., 2003.
Gambogi, J. E.; L'Esperance, R. P.; Lehmann, K. K.; Pate, B. H.; Scoles, G. (1993) J. Chem. Phys. 98, 1116-1122.
Heys, J. R. (1987) J. Chromatogr. 407, 37-47.
Hibasami, H., Borchardt, R. T., Chen, S. Y., Coward, J. K., and Pegg A. E. (1980) Studies of inhibition of rat spermidine synthase and spermine synthase, Biochem. J. 187, 419-428.
Horenstein, B. A., and Schramm, V. L. (1993) Correlation of the molecular electrostatic potential surface of an enzymatic transition state with novel transition-state inhibitors, Biochemistry 32, 9917-9925.
Kline, P. C., and Schramm, V. L. (1993) Purine nucleoside phosphorylase. Catalytic mechanism and transition-state analysis of the arsenolysis reaction, Biochemistry 32, 13212-13219.
Lee, J. E., Cornell, K. A., Riscoe, M. K., and Howell, P. L. (2001) Expression, purification, crystallization and preliminary X-ray analysis of Escherichia coli 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase, Acta Crystallogr. D. Biol. Cyystallogr. 57, 150-152.
Lee, J. E., Cornell, K. A., Riscoe, M. K., and Howell, P. L. (2003) Structure of Escherichia coli 5′-methylthioadenosine/S-adenosylhomocystein nucleosidase inhibitor complexes provide insight into the conformational changes required for substrate binding and catalysis. J. Biol. Chem. 280, 8761-8770.
Lee, J. E., Singh, V., Evans, G. B., Tyler, P. C., Furneaux, R. H., Cornell, K. A., Riscoe, M. K., Schramm V. L., and Howell P. L. (2005) Structural rationale for the affinity of pico- and femtomolar transition state analogues of E. coli 5′-methylthioadenosine/s-adenosylhomocysteine nucleosidase, J. Biol. Chem. 280, 18274-18282.
Lewandowicz, A., and Schramm, V. L. (2004) Transition state analysis for human and Plasmodium falciparum: purine nucleoside phosphorylases, Biochemistry 43, 1458-1468.
Lewandowicz, A., Tyler, P. C., Evans, G. B., Furneaux, R. H., and Schramm, V. L. (2003) Achieving the ultimate physiological goal in transition state analogue inhibitors for purine nucleoside phosphorylase. J. Biol. Chem. 278, 31465-31468.
Johnsson, T.; Edmondson, D. E.; Klinmann, J. P. (1994) Biochemistry 33, 14871-14878.
Kicska, G. A.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.; Fedorov, A.; Lewandowicz, A.; Cahill, S. M.; Almo, S. C.; Schramm, V. L. (2002) Biochemistry 41, 14489-14498.
Lewis, B. E., and Schramm, V. L. (2001a) Conformational equilibrium isotope effects in glucose by 13C NMR spectroscopy and computational studies, J. Am. Chem. Soc. 123, 1327-1336.
Lewis, B. E., and Schramm, V. L. (2001b) Binding equilibrium isotope effects for glucose at the catalytic domain of human brain hexokinase, J. Am. Chem. Soc. 125, 4785-4798.
Lewis, B. E.; Schramm, V. L. (2003a) J. Am. Chem. Soc. 125, 4785-4798.
Lewis, B. E., and Schramm, V. L. (2003b) Isotope effect-mapping of the ionization of glucose demonstrates unusual charge sharing, J Am. Chem. Soc. 125, 7872-7877.
Lewis, E. S. (1959) Tetrahedron 5, 143-148.
Lewis, E. S.; Boozer, C. E. (1952) J. Am. Chem. Soc. 74, 6306-6307.
Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm V. L. (1998) Biochemistry 37, 8615-8621.
Miller, B. G.; Wolfenden, R. (2002) Annu. Rev. Biochem. 71, 847-885.
Miller, C. H.; Duerre, J. A. (1968) J. Biol. Chem. 243, 92-97.
Miller, M. B., and Bassler, B. L. (2001) Quorum sensing in bacteria, Annu. Rev. Microbiol. 55, 165-199.
Miller, M. B., Skorupski, K., Lenz, D. H., Taylor, R. K., and Bassler, B. L. (2002) Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae, Cell 110, 303-314.
Miller, S. T., Xavier, K. B., Campagna, S. R., Taga, M. E., Semmelhack, M. F., Bassler, B. L., and Hughson, F. M. (2004) Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2. Mol. Cell. 15, 677-687.
Myers, R. W., and Abeles, R. H. (1989) Conversion of 5-S-ethyl-5-thio-D-ribose to ethionine in Klebsiella pneumoniae. Basis for the selective toxicity of 5-S-ethyl-5-thio-D-ribose, J. Biol. Chem. 264, 10547-10551.
Northcott, D.; Robertson, R. E. (1969) J. Phys. Chem. 73, 1559-1563.
Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions, Biochemistry 14, 2644-2651.
Northorp, D. B. (1981) The expression of isotope effects on enzyme-catalyzed reactions, Annu. Rev. Biochem. 50, 103-131.
Pajula, R. L., and Raina, A. (1979) Purification of spermine synthase from bovine brain by spermine-Sepharose affinity chromatography. FEBS Lett. 99, 153-156.
Paneth B. Applications of Heavy Atom Isotope Effects. In Synthesis and Applications of Isotopically Labeled Compounds 1997.
Parkin, D. W., Leung, H. B., and Schramm, V. L. (1984) Synthesis of nucleotides with specific radiolabels in ribose. Primary 14C and secondary 3H kinetic isotope effects on acid-catalyzed glycosidic bond hydrolysis of AMP, dAMP, and inosine, J. Biol. Chem. 259, 9411-9417.
Parsek, M. R., Val, D. L., Hanzelka, B. L., Cronan, J. E. Jr., and Greenberg, E. P. (1999) Acyl homoserine-lactone quorum-sensing signal generation, Proc. Natl. Acad. Sci. USA 96, 4360-4365.
Pegg, A. E. (1983) Inhibition of Aminopropyltransferases. Methods Enzymol. 94, 294-297.
Pham, T. V., Fang, Y. R., and Westaway, K. C. (1997) Transition state looseness and α-secondary kinetic isotope effects. J. Am. Chem. Soc. 119, 227-232.
Ragione, D.; Porcelli, F. M.; Carteni-Farina, M.; Zappia, V.; and Pegg, A. E. (1985) Biochem. J. 232, 335-341.
Riscoe, M. K.; Ferro, A. J.; Fitchen, J. H. (1989) Parasitol. Today 5, 330-333.
Rising, K. A., and Schramm V. L. (1994) Enzymatic synthesis of NAD+ with the specific incorporation of atomic labels, J. Am. Chem. Soc. 116, 6531-6536.
Rose, I. A. (1980) The isotope trapping method: desorption rates of productive E.S complexes, Methods Enzymol. 64, 47-59.
Sauve, A. A., Cahill, S. M., Zech, S. G., Basso, L. A., Lewandowicz, A., Santos, D. S., Grubenmeyer, C., Evans, G. B., Fumeaux. R. H., Basso, L. A., Santos, D. S., Almo, S. C., and Schramm, V. L. (2003) Ionic states of substrates and transition state analogues at the catalytic sites of N-ribosyltransferases. Biochemistry 42, 5694-5705.
Schauder, S., Shokat, K., Surette, M. G., and Bassler, B. L. (2001) The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule, Mol. Microbiol. 41, 463-476.
Schramm, V. L. (2005) Enzymatic transition states: thermodynamics, dynamics and analogue design. Arch. Biochem. Biophys. 433, 13-26.
Shi, W; Basso, L. A.; Santos, D. S.; Tyler, P. C.; Furneaux, R. H.; Blanchard, J. S.; Almo, A. C.; Schramm, V. L. (2001a) Biochemistry 40, 8204-8215.
Shi, W., Tanaka, K. S. E., Crother, T. R., Taylor, M. W., Almo, S. C., and Schramm, V. L. (2001B) Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae, Biochemisty 40, 10800-10809.
Shiner, V. J., Jr. (1959) Tetrahedron 5, 243-252.
Singh, V.; Shi, W.; Evans, G. B.; Tyler, P. C.; Furneaux, R. H.; Schramm, V. L. (2004) Biochemistry 43, 9-18.
Singh, V.; Lee, J. E.; Nunez, S.; Howell, L. P.; Schramm, V. L. (2005a) Biochemistry 44, 11647-11659.
Singh, V., Evans, G. B., Lenz, D. H., Mason, J. M., Clinch, K., Mee, S., Painter, G. F., Tyler, P., C., Furneaux, R. H., Lee, J. E., Howell, P. L., and Schramm, V. L., (2005b) Femtomolar transition state analogue inhibitors of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli. J. Biol. Chem. 280, 18265-18273.
Singh, V.; Shi, W.; Almo, S. C.; Evans, G. B.; Furneaux, R. H.; Tyler, P. C.; Zheng, R.; Schramm, V. L. (2006) Biochemistry 2006, submitted.
Sufrin, J. R.; Meshnick, S. R.; Spiess, A. J.; Garofalo-Hanuman, J.; Pan, X, Q.; Bacchi, C. J. (1995) Antimicrob. Agents Chemother. 39, 2511-2515.
Sunko, D. E., Szele, I., and Hehre, W. J. (1997) Hyperconjugation and the angular dependence of .beta.-deuterium isotope effects, J. Am. Chem. Soc. 99, 5000-5005.
Tabor, C. W.; Tabor, H. (1983) Methods Enzymol. 94, 294-297
Williams-Ashman, H. G., Seidenfeld, J., and Galletti, P. (1982) Trends in the biochemical pharmacology of 5′-deoxy-5′-methylthioadenosine, Biochem. Pharmacol. 31, 277-288.
Winzer, K., Hardie, K. R., Burgess, N., Doherty, N., Kirke, D., Holden, M. T., Linforth, R., Cornell, K. A., Taylor, A. J., Hill, P. J., and Williams, P. (2002) LuxS: its role in central metabolism and the in vitro synthesis of 4-hydroxy-5-methyl-3(2H)-furanone, Microbiology 148, 909-922.
Withers, H.; Swift, H. S.; Williams P. (2001) Curr. Opin. Microbiol. 4, 186-193.
Wolfenden, R. (1969) Transition state analogues for enzyme catalysis. Nature 223, 704-705.
Wolfenden, R., and Snider, M. J. (2001) The depth of chemical time and the power of enzymes as catalysts, Acc. Chem. Res. 34, 938-945.
Xavier, K. B.; Bassler B. L. (2003) Curr. Opin. Microbial. Rev. 6, 191-197.
Xue, Q.; Horsewill, A. J.; Johnson, M. R.; Trommsdorff, H. P. (2004) J. Chem. Phys. 120, 1107-11119.
Zhou, G. C., Parikh, S. L., Tyler, P. C., Evans, G. B., Furneaux, R. H., Zubkova, O. V., Benjes, P. A., and Schramm, V. L. (2004) Inhibitors of ADP-ribosylating bacterial toxins based on oxacarbenium ion character at their transition states, J. Am. Chem. Soc. 126, 5690-5698.
Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) plays an important role in biological processes including polyamine biosynthesis, methylation, purine salvage and quorum sensing (Hibasami et al., 1980; Carteni-Farina et al., 1983; Winzer et al., 2002; Chen et al., 2002; Williams-Ashman et al., 1982; Miller and Bassler, 2001; Miller at al., 2002). It catalyzes the physiologically irreversible hydrolytic depurination of 5′-methylthioadenosine (MTA) to generate adenine and 5-methylthioribose (FIG. 1A). Adenine is salvaged by adenine phosphoribosyltransferase and the 5-methylthioribose (MTR) can be recovered in the methionine salvage pathway (Myers and Abels, 1989). In E. coli, MTA is generated as a by-product of polyamine synthesis from the reaction involving transfer of the aminopropyl group from decarboxylated SAM to putresine. Spermidine synthase is reported to be sensitive to product inhibition by MTA with the inhibition constant of 50 μM for rat spermidine synthase (Pegg, 1983). Mammalian spermine synthase is more sensitive to MTA, with a Ki value of 0.3 μM (Pajula and Raina, 1979). Inhibition of MTAN is therefore expected to inhibit polyamine biosynthesis and the salvage pathways for adenine and methionine. In bacteria MTA is also produced as a by-product in the synthesis of acyl homoserine lactones (AHL) from S-adenosylmethionine (SAM) and acyl-ACP in a reaction catalyzed by AHL synthase (Parsek et al., 1999). Acylhomoserine lactones are also known as autoinducers-1 (AI1) and are used by gram negative bacteria for quorum sensing (FIG. 1B). MTA has an inhibitory effect on AHL synthase (50 μM produces 67% inhibition) (Parsek et al., 1999). In addition to MTA, MTAN also catalyzes hydrolysis of S-adenosylhomocysteine (SAH) to generate adenine and S-ribosylhomocysteine (SRH). SRH is subsequently converted to a group of furanone-like molecules that are collectively known as autoinducer-2 (AI2) (one example is shown in FIG. 1B) (Schauder et al., 2001). Autoinducers (AI1 and AI2) mediate quorum sensing in bacteria to regulate processes such as biofilm formation, virulence and antibiotic resistance. Disruption of these pathways by inhibiting MTAN presents a potential target for interfering with biofilm formation and autoinducer-mediated antibiotic resistance pathways.
Transition state theory predicts that enzymes catalyze reactions by lowering the activation barrier and the catalytic acceleration imposed by the enzyme is proportional to the enzymatic stabilization of the transition state (Wolfe) den, 1969; Wolgenden and Snider, 2001). Transition state analogue inhibitors are designed from the hypothesis that chemically stable analogues that mimic geometric and molecular electrostatic features of the transition state will bind to enzyme tighter than the substrate by a factor approaching the catalytic rate acceleration imposed by the enzyme. For nucleoside hydrolases the calculation predicts a binding affinity of 10−19 to 10−18M for mimics of the transition state (DeWolf et al., 1979; Horenstein and Schramm, 1993; Cornell et al., 1996). However, it is not possible to design “perfect” transition state analogues since the actual enzymatic transition state involves non-equilibrium bond lengths and charges that cannot be accurately copied to chemically stable molecules.
Kinetic isotope effects (KIEs) using isotopically labeled substrates combined with computational chemistry are the preferred method to understand the transition states of enzymatic reactions (Lewandowicz and Schramm, 2004; Chen et al., 2000; Birck and Schramm, 2004a; Schramm, 2005). KIEs are defined as the ratio of reaction rates for normal and isotopically labeled substrate. Competitive KIEs measure the effect on kcat/Km which includes all steps from free reactants to the first irreversible step of the reaction. Intrinsic isotope effects occur when the first irreversible step is bond breaking at the transition state and none of the intervening steps present a significant energetic barrier. Intrinsic KIEs report the difference between bond vibrational ground states for the reactants free in solution and at the transition state. Computational modeling of transition states is facilitated by using intrinsic KIEs as experimental boundary conditions.
Kinetic isotope effects have been used to study the transition states of N-ribosyltransferases including nucleoside hydrolase, purine nucleosidase phosphorylase (PNP), ricin-A chain and thymidine phosphorylase (Horenstein and Schramm, 1993; Lewandowicz and Schramm, 2004; Chen et al., 2000; Birck and Schramm, 2004a). Most N-ribosyltransferases (with the exception of thymidine phosphorylase) have dissociative SN1 reaction mechanisms with transition states exhibiting ribooxacarbenium ion character. Transition state analogue inhibitors have been designed for some of these enzymes by incorporating properties of their transition states into chemically stable analogues. They are powerful inhibitors. One such example is Immucillin-H, a transition state analogue inhibitor of human and bovine PNPs that binds with dissociation constants of 56 pM and 23 pM, respectively. It is currently in clinical trials for T-cell leukemia under the name Fodosine3. Second-generation transition state analogues designed specifically to match the transition state of human PNP are tight-binding inhibitors with Kd values to 7 pM and one of these is in clinical trials for psoriasis (Lewandowicz et al., 2003). See http://www.biocryst.com for clinical trial information.